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Comparison of methods for extracting thylakoid membranes of Arabidopsis plants
Umeå University, Faculty of Science and Technology, Department of Chemistry. College of Life Sciences, Sichuan Agricultural University, Ya'an, China.
Umeå University, Faculty of Science and Technology, Department of Chemistry.
2016 (English)In: Physiologia Plantarum: An International Journal for Plant Biology, ISSN 0031-9317, E-ISSN 1399-3054, Vol. 156, no 1, 3-12 p.Article in journal (Refereed) Published
Abstract [en]

Robust and reproducible methods for extracting thylakoid membranes are required for the analysis of photosynthetic processes in higher plants such as Arabidopsis. Here, we compare three methods for thylakoid extraction using two different buffers. Method I involves homogenizing the plant material witha metal/glass blender; method II involves manually grinding the plant materialin ice-cold grinding buffer with a mortar and method III entails snap-freezing followed by manual grinding with a mortar, after which the frozen powder is thawed in isolation buffer. Thylakoid membrane samples extracted using each method were analyzed with respect to protein and chlorophyll content, yields relative to starting material, oxygen-evolving activity, protein complex content and phosphorylation. We also examined how the use of fresh and frozen thylakoid material affected the extracts’ contents of protein complexes. The use of different extraction buffers did not significantly alter the protein contentof the extracts in any case. Method I yielded thylakoid membranes with the highest purity and oxygen-evolving activity. Method III used low amounts of starting material and was capable of capturing rapid phosphorylation changes in the sample at the cost of higher levels of contamination. Method II yielded thylakoid membrane extracts with properties intermediate between those obtained with the other two methods. Finally, frozen and freshly isolated thylakoid membranes performed identically in blue native-polyacrylamide gel electrophoresis experiments conducted in order to separate multimeric protein supracomplexes.

Place, publisher, year, edition, pages
2016. Vol. 156, no 1, 3-12 p.
Keyword [en]
BN-PAGE, blue native-polyacrylamide gel electrophoresis, Chl, chlorophyll, COXII, cytochrome oxidase subunit II, LHC, light-harvesting complex, PS, photosystem, SDS, sodium dodecyl sulfate
National Category
Analytical Chemistry Plant Biotechnology
Identifiers
URN: urn:nbn:se:umu:diva-110442DOI: 10.1111/ppl.12384ISI: 000367687800002PubMedID: 26337850OAI: oai:DiVA.org:umu-110442DiVA: diva2:862292
Available from: 2015-10-21 Created: 2015-10-21 Last updated: 2016-03-11Bibliographically approved

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Chen, Yang-ErSchröder, Wolfgang P.
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Physiologia Plantarum: An International Journal for Plant Biology
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